The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of 125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of 125I-001-C8 (7.24 +/- 0. 41 microl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 microM), protamine (1 mM), poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-L-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of 125I-001-C8 were 1.13 +/- 0.23 pmol/mg protein, 0. 47 +/- 0.43 microM, and 3.13 +/- 0.19 microl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.